Metabolic syndrome (MetS) is associated with an increased risk of stroke and poor outcome, yet the mechanisms underlying this health predicament are unclear. Hypertension, a revolving complication of MetS, disrupts cerebral vasoregulatory mechanisms and increases the susceptibility of the brain to ischemic injury, is more prevalent in postmenopausal women. Emerging evidence suggests that patients with MetS are associated with poor collateral status during acute ischemic stroke. Our recent data showed that female mice with MetS exhibited poor collateral status during stroke and larger stroke size after MCAO. Mice with MetS also have reduced blood flow in penetrating arterioles and impairment in microvascular flow dynamics. Ample evidence links diabetes to impaired vasodilation that is attributable to reduced eNOS phosphorylation; however, the role of eNOS in mediating pial and penetrating arteriole flow after stroke has never been properly addressed. Given the known effect of estrogen on increasing eNOS activity and the fact that T2DM associated reduction in estrogen, it is unclear whether the impaired collateral flow in the aged female mice with MetS is attributed to reduced eNOS activity. Clinical evidence also suggests that MetS induced-hypercoagulability has a greater impact on adverse vascular events in women than in men including the risk of ischemic stroke. Extracellular vesicles (EV) and exosomes are recognized as key players in cardiovascular and metabolic diseases, and platelet-derived EVs are particularly important in mediating thrombotic events that can potentiate the risk of stroke and worsen blood flow. The proposed study will investigate the causal relationship between reduced blood perfusion and impaired eNOS as well as EV-mediated increase in platelet activation/coagulation in reproductively senescent female mice with MetS. Cutting-edge technology including Doppler Optical Coherence Tomography and Multiphoton Laser Scanning Microscopy will be employed to detect blood flow dynamics in pial and penetrating arterioles as well as capillary vessels, clot and platelet-leukocyte aggregates formation before and after stroke. Transgenic knock-in mice expressing phosphomimetic eNOS will be used to ascertain the role of vasodilation in regulating collateral flow in db/db females. The role of EVs in increasing clotting and causing collateral failure will be tested in vivo by injecting Isolated EVs into the jugular vein of MetS and normal mice. The proposed studies in this application will provide insights into the complex biology of poor blood perfusion especially in women with MetS with proof-of-concept data for future development of collateral therapy.